Reversible palmitoylation is catalyzed by palmitoyltransferase(PAT) from the zinc-finger DHHC domain-containing protein (ZDHHC) family. The Zdhhc19 knockout male mice showed infertility and Zdhhc19 deficient sperm showed severe morphological defects. To identify the candidate substrates of ZDHHC19 in haploid spermatids, we carried out label free mass spectrometry (MS) combined with click chemistry. This study reveals the role of palmitoylation mediated by ZDHHC19 during spermiogenesis.